The invention relates generally to methods of detecting prions in samples such as blood or tissue.
Prions are infectious pathogens that cause central nervous system spongiform encephalopathies in humans and animals. Prions are distinct from bacteria, viruses and viroids. The predominant hypothesis at present is that no nucleic acid component is necessary for infectivity of prion protein. Further, a prion which infects a first species of animal (e.g., a human) will not infect a second species which is genetically diverse form the first species (e.g., a mouse).
A major step in the study of prions and the diseases that they cause was the discovery and purification of a protein designated prion protein (xe2x80x9cPrPxe2x80x9d) (Bolton et al., Science 218:1309-11 (1982); Prusiner et al., Biochemistry 21:6942-50 (1982); McKinley et al., Cell 35:57-62 (1983)). Complete prion protein-encoding genes have since been cloned, sequenced and expressed in transgenic animals. PrPC is encoded by a single-copy host gene (Basler et al., Cell 46:417-28 (1986)) and is normally found at the outer surface of neurons. During a post-translational process, PrPSc is formed from the normal, cellular PrP isoform (PrPC), and prion diseases result from conversion of PrPC into a modified isoform called PrPSc. PrPSc is necessary for both the transmission and pathogenesis of the transmissible neurodegenerative diseases of animals and humans.
See Prusiner, S. B., Science 252:1515-1522 (1991). The most common prion diseases of animals are scrapie of sheep and goats, chronic wasting disease of deer and elk, and bovine spongiform encephalopathy (BSE) of cattle (Wilesmith, J. and Wells, Microbiol. Immunol. 172:21-38 (1991)). Four prion diseases of humans have been identified: (1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3) Gerstmann-Strassler-Scheinker Disease (GSS), and (4) fatal familial insomnia (FFI) (Gajdusek, D. C., Science 197:943-960 (1977); Medori et al., N. Engl. J Med. 326:444-449 (1992)). The presentation of human prion diseases as sporadic, genetic and infectious illnesses initially posed a conundrum which has been explained by the cellular genetic origin of PrP.
Variations in prions, which cause different disease phenotypes, are often referred to as strains. Each prion strain produces a specific phenotype of prion disease as manifested by the length of the incubation time, the topology of PrPSc accumulation, and the distribution of pathological lesions (Fraser and Dickinson 1968; Fraser and Dickinson 1973; Bruce, McBride et al. 1989; Taraboulos, Jendroska et al. 1992; DeArmond, Yang et al. 1993; Scott, Groth et al. 1997). Strains replicate with a high degree of fidelity, which demands a mechanism that can account for this phenomenon. That strains could be accounted for because PrPSc might exist in multiple conformations was postulated, but supporting evidence was initially lacking (Prusiner 1991; Cohen, Pan et al. 1994). Subsequently, different lines of investigation, one on the isolation of prion strains from mink by passage in hamsters (Bessen and Marsh 1994) and the other on the passage of inherited human prion diseases to transgenic (Tg) mice (Telling, Parchi et al. 1996), converged to argue that the properties of prion strains are enciphered in the conformation of PrPSc.
The detection of PrPSc in biological products is of critical importance, as prion diseases are transmissible. Iatrogenic CJD has been caused by human growth hormone derived from cadaveric pituitaries as well as dura mater grafts (Brown et al., Lancet 340:24-27 (1992)). In addition, kuru, which for many decades devastated the Fore and neighboring tribes of the New Guinea highlands, is believed to have been spread by infection during ritualistic cannibalism (Alpers, M. P., Slow Transmissible Diseases of the Nervous System Vol. 1, S. B. Prusiner and W. J. Hadlow, eds. (New York: Academic Press), pp. 66-90 (1979)). Numerous young adults treated with HGH derived from human pituitaries have developed CJD (Koch et al., N. Engl. J Med. 313:731-733 (1985); Brown et al., Lancet 340:24-27 (1992); Fradkin et al., JAMA 265:880-884 (1991); Buchanan et al., Br. Med. J. 302:824-828 (1991)). In addition, hundreds of children in France have been treated with growth hormone extracted from dead bodies at the risk of developing CJD (see New Scientist, Nov. 20, 1993, page 10.) That the HGH prepared from pituitaries was contaminated with prions is supported by the transmission of prion disease to a monkey 66 months after inoculation with a suspect lot of HGH (Gibbs, Jr. et al., N. Engl. J. Med. 328:358-359 (1993)).
The long incubation times associated with prion diseases will not reveal the full extent of iatrogenic CJD for decades in thousands of people treated with HGH worldwide. Iatrogenic CJD also appears to have developed in four infertile women treated with contaminated human pituitary-derived gonadotrophin hormone (Healy et al., Br. J. Med. 307:517-518 (1993); Cochius et al., Aust. N. Z. J. Med. 20:592-593 (1990); Cochius et al., J. Neurol. Neurosurg. Psychiatry 55:1094-1095 (1992)) as well as at least 11 patients receiving dura mater grafts (Nisbet et al., J. Am. Med. Assoc. 261:1118 (1989); Thadani et al., J. Neurosurg. 69:766-769 (1988); Willison et al., J. Neurosurg. Psychiatric 54:940 (1991); Brown et al., Lancet 340:24-27 (1992)). These cases of iatrogenic CJD underscore the need for screening pharmaceuticals and biological products that might possibly be contaminated with prions.
The importance of detecting prions in biological products has been heightened by the possibility that bovine prions have been transmitted to humans who developed new variant Creutzfeldt-Jakob disease (nvCJD) (G. Chazot et al., Lancet 347:1181 (1996); R. G. Will et al. Lancet 347:921-925 (1996)). Earlier studies had shown that the N-terminus of PrPSc could be truncated without loss of Scrapie infectivity (S. B. Prusiner et al., Biochemistry 21:6942-6950 (1982); S. B. Prusiner et al., Cell 38:127-134 (1984)) and correspondingly, the truncation of the N-terminus of PrPSc still allowed its conversion into PrPSc (M. Rogers et al., Proc. Natl. Acad. Sci. USA 90:3182-3186 (1993)). The ability of transmission of nvCJD from cattle to humans has been confirmed through in vivo testing, suggesting that the December 20 issue of Proceedings of National Academy of Sciences undermining the comforting presumption that the documented xe2x80x9cspecies barrierxe2x80x9d is relevant to this new strain (M. R. Scott et al., Proc. Natl. Acad. Sci. USA 96:15137-15142 (1999)).
In view of such, there clearly is a need for a convenient, cost-effective means for identifying prions which cause CJD in biological products, e.g., blood and blood products.
The present invention provides assays that can identify the levels of both protease sensitive and protease resistant conformers of PrPSc in a sample. In a preferred embodiment, the assay comprises the steps of 1) determining levels of total PrPSc in a sample; 2) subjecting the PrPSc fraction to treatment with a protease that selectively hydrolzes the protease sensitive PrPSc (sPrPSc) conformers and 3) quantifying the levels of sPrPSc in the sample. The ability to detect sPrPSc allows early detection of prions, since the PrPSc in easily accessible biological samples such as blood is predominantly sPrPSc and not protease resistant PrPSc, i.e., not rPrPSc. These assay can identify prion infection in a sample prior to the accumulation of the protease resistant PrPSc (rPrPSc) conformers to a level associated with classic symptoms of prison infection. The present invention thus provides an improved method of early detection of these infectious agents in products such as human biologicals and food.
Methods of the present invention also have the ability to distinguish between different prion isolates (xe2x80x9cstrainsxe2x80x9d), and thus can identify a specific prion strain in an infected sample. The ratio of rPrPSc to sPrPSc remains relatively constant for each particular strain during prion infection. By determining the levels of rPrPSc and calculating a ratio rPrPSc levels to the levels of sPrPSc, a specific prion strain can be identified based on the ratio of in vivo levels of these two PrPSc conformers.
In one embodiment of the invention, the total PrPSc fraction is isolated prior to treatment, e.g, by exposing a sample to a complexing agent which selectively binds to PrPSc, e.g., selectively binds to both rPrPSc and sPrPSc.
In a particular embodiment, the PrPSc conformers are identified using limited proteinase K digestion followed by indentification by an antibody which recognizes rPrPSc or sPrPSc, but not both. For example, the concentration of total PrPSc is measured in a sample by comparing the concentration of PrPSc to PrPC following limited proteolysis. A second aliquot of a sample is then completely digested (i.e. to remove PrPC and relax the conformation of sPrPSc) and the amount of rPrPSc measured. The amount of sPrPSc in a sample can then be determined by substracting the concentration of rPrPSc from the concentration of total PrPSc.
It is an object of the invention to provide a specific diagnostic assay for prion infection in variable sample material obtained or derived from human, primate, monkey, hamster, mice, pig, bovine, sheep, deer, elk, cat, dog, and chicken tissues.
It is another object of the invention to provide a strain-specific diagnostic and/or prognostic assay for prion infection.
It is yet another object of the invention to identify PrPSc in the pre-symptomatic stage when proteinase K-resistant forms of PrPSc are not yet present in phenotypically detectable quantities.
It is yet another object of the invention to provide the disease-specific diagnostic assay for prion infection in the tissues, where the concentration of rPrPSc is below detectable levels or may not accumulate at all. Examples of such samples are serum and white blood cells (WBC) isolated from prion-infected whole blood.
It is yet another object of the invention to provide a rapid assay to differentiate prion strains in prion-infected animals specifically by determining the ratio of sPrPSc to rPrPSc in a sample taken from an infected animal.
One advantage of the present methods is that they are rapid, reliable, and require relatively few reagents or equipment.
An important feature of the assay is the rapid, cost-effective, and high flow-through design with capacity to screen a high volume of samples.
These and other objects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the methods as more fully described below.